This proposal brings together an interdisciplinary team of collaborators to address a major challenge: to achieve clinical utility of patient-specific induced pluripotent stem cells (iPSCs) for blood diseases. Our ultimate goal is to differentiate customized iPSCs into hematopoietic stem and progenitor cells to accurately model human blood diseases for research into disease mechanisms, and as a platform for treating patients with blood diseases. To achieve this, this proposal aims to discover the developmental pathways and biomechanical principles that drive the formation of hematopoietic stem cells (HSCs) in embryos, and will screen for morphogens and chemicals that promote hematopoietic maturation in vitro. We will take several novel and integrated approaches including: 1) application of predictive computational algorithms to gene expression data from highly purified embryonic HSC populations to discover the gene networks that direct hematopoietic and lymphoid development; 2) Screens in zebrafish embryos and murine and human pluripotent stem cells to discover novel chemical and biological regulators of HSCs; 3) bioengineered platforms for production of hematopoietic stem and progenitor populations, applying biomechanical forces to mimic the embryonic microenvironment; 4) derivation of iPSC from patients with Primary Immune Deficiency to correlate genotype with lymphoid phenotypes and to test strategies for gene repair through an in vitro clinical trial; and ultimately, 5) to derive transgene-free clinical-grade pluripotent stem cells, and develop protocols for differentiation of hematopoietic populations at clinical scale. Success in generating engraftable, genetically unperturbed HSC would be a significant breakthrough that would enhance the utility of iPSC for modeling hematopoietic disease and establish a translational platform for combining gene repair with HSC transplantation therapy.